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J Vis Exp. 2018 Nov 1;(141). doi: 10.3791/58162.

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney.

Author information

1
Division of Renal-Electrolyte, Department of Medicine, University of Pittsburgh.
2
Department of Cell Biology, University of Pittsburgh.
3
Division of Renal-Electrolyte, Department of Medicine, University of Pittsburgh; tanrj@upmc.edu.

Abstract

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

PMID:
30451231
PMCID:
PMC6688477
DOI:
10.3791/58162
[Indexed for MEDLINE]
Free PMC Article

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